1. Field of the Invention
The present invention is directed to a method of in vivo and ex vivo gene delivery, for a variety of cells. More specifically, it relates to a novel carrier system and method for targeted delivery of nucleic acids to mammalian cells. More specifically, the present invention relates to carrier systems comprising single-chain polypeptide binding molecules having a basic amino acid rich region, such as an oligo-lysine or an oligo-arginine region, and having the three dimensional folding and, thus, the binding ability and specificity, of the variable region of an antibody. Such preparations of modified single chain polypeptide binding molecules also have ability to bind nucleic acids at the basic amino acid rich region. These properties of the modified single chain polypeptide binding molecules make them very useful in a variety of therapeutic applications including gene therapy. The invention also relates to multivalent antigen-binding molecules having basic amino acid rich regions. Compositions of, genetic constructions for, methods of use, and methods for producing such basic amino acid rich region containing antigen-binding proteins are disclosed.
2. Background Art
Substantial attention has been given to the promise of gene therapy in recent years. This term has been used to describe a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. Such methods include, for example, the delivery of a gene, antisense RNA, a cytotoxic agent, etc., by a vector to a mammalian cell, preferably a human cell either in vivo or ex vivo. Most of the initial work has focused on the use of retroviral vectors to transform these cells. This focus has resulted from the ability of retroviruses to infect cells with high efficiency.
However, numerous difficulties with retroviruses have been reported. For example, problems have been encountered in infecting certain cell types. Retroviruses typically enter cells via receptors and if such receptors are not present on the cell, or not present in large numbers, then infection is not possible or efficient. These viruses are also relatively labile in comparison to other viruses. Outbreaks of wild-type virus from recombinant virus-producing cell lines have also been reported with the vectors themselves causing disease. Moreover, these viruses are only expressed in dividing cells.
In addition, retroviral-mediated gene transfer methods typically result in stable transformation of the target cells. Although this may be regarded as advantageous, the stable transformation of a patient""s somatic cells makes it difficult to reverse the treatment regimen if undesirable side effects occur. Moreover, there is the concern that genetic transformation might lead to malignant transformation of the cell.
Other methods of delivering genetic material to cells in vivo and ex vivo include the use of liposome entrapped DNA. Liposomes are small membrane-enclosed spheres that have been formed with the appropriate DNA entrapped within it. However, this system also has inherent problems. It is difficult to control the size of the liposome and, hence the uniformity of delivery to individual cells. Additionally, it is difficult to prevent leakage of the contents of the liposomes and as with other techniques, there has been difficulty in directing cell-type specificity.
Antibodies are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule, termed an antigen. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule xe2x80x9crecognizesxe2x80x9d the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
The antibody molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. The remainder of this discussion on antibodies will refer only to one pair of light/heavy chains, as each light/heavy pair is identical. Each individual light and heavy chain folds into regions of approximately 110 amino acids, assuming a conserved three-dimensional conformation. The light chain comprises one variable region (VL) and one constant region (CL), while the heavy chain comprises one variable region (VH) and three constant regions (CH1, CH2 and CH3). Pairs of regions associate to form discrete structures. In particular, the light and heavy chain variable regions associate to form an xe2x80x9cFvxe2x80x9d area which contains the antigen-binding site. The constant regions are not necessary for antigen binding and in some cases can be separated from the antibody molecule by proteolysis, yielding biologically active (i.e., binding) variable regions composed of half of a light chain and one quarter of a heavy chain.
Further, all antibodies of a certain class and their Fab fragments (i.e., fragments composed of VL, CL, VH, and CH1) whose structures have been determined by x-ray crystallography show similar variable region structures despite large differences in the sequence of hypervariable segments even when from different animal species. The immunoglobulin variable region seems to be tolerant towards mutations in the antigen-binding loops. Therefore, other than in the hypervariable regions, most of the so-called xe2x80x9cvariablexe2x80x9d regions of antibodies, which are defined by both heavy and light chains, are, in fact, quite constant in their three dimensional arrangement. See for example, Huber, R., Science 233:702-703 (1986).
Recent advances in immunobiology, recombinant DNA technology, and computer science have allowed the creation of single polypeptide chain molecules that bind antigen. These single-chain antigen-binding molecules (xe2x80x9cSCAxe2x80x9d) or single-chain variable fragments of antibodies (xe2x80x9csFvxe2x80x9d) incorporate a linker polypeptide to bridge the individual variable regions, VL and VH, into a single polypeptide chain. A description of the theory and production of single-chain antigen-binding proteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,455,030 and 5,518,889. The single-chain antigen-binding proteins produced under the process recited in the above U.S. patents have binding specificity and affinity substantially similar to that of the corresponding Fab fragment. A computer-assisted method for linker design is described more particularly in Ladner et al., U.S. Pat. Nos. 4,704,692 and 4,881,175, and WO 94/12520.
The in vivo properties of sFv polypeptides are different from MAbs and antibody fragments. Due to their small size, sFv polypeptides clear more rapidly from the blood and penetrate more rapidly into tissues (Milenic, D. E. et al., Cancer Research 51:6363-6371 (1991); Colcher et al., J. Natl. Cancer Inst. 82:1191 (1990); Yokota et al., Cancer Research 52:3402 (1992)). Due to lack of constant regions, sFv polypeptides are not retained in tissues such as the liver and kidneys. Due to the rapid clearance and lack of constant regions, sFv polypeptides will have low immunogenicity. Thus, sFv polypeptides have applications in cancer diagnosis and therapy, where rapid tissue penetration and clearance, and ease of microbial production are advantageous.
A multivalent antigen-binding protein has more than one antigen-binding site. A multivalent antigen-binding protein comprises two or more single-chain protein molecules. Enhanced binding activity, di- and multi-specific binding, and other novel uses of multivalent antigen-binding proteins have been demonstrated. See, Whitlow, M., et al., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R., Nature Biotech. 15:125-126 (1997); and WO 93/11161.
Ladner et al. also discloses the use of the single chain antigen binding molecules in diagnostics, therapeutics, in vivo and in vitro imaging, purifications, and biosensors. The use of the single chain antigen binding molecules in immobilized form, or in detectably labeled forms is also disclosed, as well as conjugates of the single chain antigen binding molecules with therapeutic agents, such as drugs or specific toxins, for delivery to a specific site in an animal, such as a human patient.
Whitlow et al. (Methods: A Companion to Methods in Enzymology 2(2):97-105 (June, 1991)) provide a good review of the art of single chain antigen binding molecules and describe a process for making them.
In U.S. Pat. No. 5,091,513, Huston et al. discloses a family of synthetic proteins having affinity for preselected antigens. The contents of U.S. Pat. No. 5,091,513 are incorporated by reference herein. The proteins are characterized by one or more sequences of amino acids constituting a region that behaves as a biosynthetic antibody binding site (BABS). The sites comprise (1) noncovalently associated or disulfide bonded synthetic VH and VL regions, (2) VH-VL or VL-VH single chains wherein the VH and VL are attached to a polypeptide linker, or (3) individual VH or VL domains. The binding domains comprises complementarity determining regions (CDRs) linked to framework regions (FRs), which can be derived from separate immunoglobulins.
U.S. Pat. No. 5,091,513 also discloses that three subregions (the CDRs) of the variable domain of each of the heavy and light chains of native immunoglobulin molecules collectively are responsible for antigen recognition and binding. These CDRs consist of one of the hypervariable regions or loops and of selected amino acids or amino acid sequences disposed in the framework regions that flank that particular hypervariable region. It is said that framework regions from diverse species are effective in maintaining CDRs from diverse other species in proper conformation so as to achieve true immunochemical binding properties in a biosynthetic protein.
U.S. Pat. No. 5,091,513 includes a description of a chimeric polypeptide that is a single chain composite polypeptide comprising a complete antibody binding site. This single chain composite polypeptide is described as having a structure patterned after tandem VH and VL domains, with a carboxyl terminal of one attached through an amino acid sequence to the amino terminal of the other. It thus comprises an amino acid sequence that is homologous to a portion of the variable region of an immunoglobulin heavy chain (VH) peptide bonded to a second amino acid sequence that was homologous to a portion of the variable region of an immunoglobulin light chain (VL)
Chen et al., describe the production and use of a fusion protein consisting of an antibody Fab fragment and a DNA binding moiety, protamine, to deliver toxin-expressing plasmid DNA into HIV infected cells by receptor mediated endocytosis (S-Y Chen et al., Gene Therapy 2: 116-123 (1995)).
Accordingly, it is an object of the present invention to provide a new and improved delivery system that can introduce foreign genes in a non-toxic, cell specific manner into mammalian cells. Also provided by the invention is a system and an efficient method that exhibits a high degree of cell specificity using relatively simple yet reliable delivery.
Another feature of the present invention is the use of receptor-mediated specificity to provide cell specificity to the gene delivery system. This involves the use of cell-surface receptors as naturally existing entry mechanisms for the specific delivery of genes. The molecules once recognized and bound to the receptor can be internalized within the target cell via endocytosis. Included in this feature is the provision for a unique carrier comprising a single-chain antigen-binding protein/polynucleotide complex capable of targeting the gene to specific cells possessing particular receptors that recognize the complex.
In addition, the carrier of the present invention relates to tailed single chain polypeptides containing a basic amino acid rich region (i.e., oligo-lysine, oligo-arginine, or a mixture thereof) and having binding affinity for an antigen and the capability of delivering nucleic acids to a cell and processes for preparing them. Suitable polypeptides are, for example, those described by Ladner et al. in U.S. Pat. No. 4,946,778 and Huston et al. in U.S. Pat. No. 5,091,513.
These features provide advantages to the present invention that directly contribute to the efficiency and target specificity of the delivery system to specific cell types, including normal cells as well as tumor cells not found in the delivery systems known in the art.
The present invention is directed to a method of delivering nucleic acids to a cell comprising:
(1) providing an a basic amino acid tailed single-chain antigen-binding polypeptide capable of delivering nucleic acids to a cell comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein, at its C-terminus, N-terminus, or both of polypeptide (a), (b) or both, the single-chain antigen-binding polypeptide has an amount of basic amino acid residues sufficient to bind nucleic acids, wherein the basic amino acid residues are selected from the group consisting of: Lys, Arg and a combination thereof; and
wherein the basic amino acid residues binds nucleic acid and wherein the single-chain antigen-binding polypeptide binds antigen;
(2) allowing a nucleic acid to bind to the basic amino acid residue containing single-chain antigen-binding polypeptide; and
(3) transforming a cell with the nucleic acid bound basic amino acid residue containing single-chain antigen-binding polypeptide.
More particularly, the invention is directed to a single-chain antigen-binding polypeptide capable of delivering nucleic acids to a cell, comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b) or both, the single-chain antigen-binding polypeptide has an amount of basic amino acid residues sufficient to bind nucleic acids, wherein the basic amino acid residues are selected from the group consisting of: Lys, Arg and a combination thereof; and wherein the basic amino acid residues binds nucleic acid and wherein the single-chain antigen-binding polypeptide binds antigen. These basic amino acid residues in the sFv protein (e.g., oligo-lysine sFv) generate a minimal non-specific nucleic acid binding region. The basic amino acid region is configured such that at least 2 to 8 groups of eight consecutive residues of Lys, Arg or a combination thereof are separated from adjacent groups by 0-20 amino acid residues.
The invention is further directed to a genetic sequence encoding a single-chain antigen-binding polypeptide capable of delivering nucleic acids to a cell, comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b) or both, the single-chain antigen-binding polypeptide has an amount of basic amino acid residues sufficient to bind nucleic acids, wherein the basic amino acid residues are selected from the group consisting of: Lys, Arg and a combination thereof; and
wherein the basic amino acid residues binds nucleic acid and wherein the single-chain antigen-binding polypeptide binds antigen. These basic amino acid residues in the sFv protein (e.g. oligo-lysine sFv) generate a minimal non-specific nucleic acid binding region. The basic amino acid region is configured such that at least 2 to 8 groups of eight consecutive residues of Lys, Arg or a combination thereof are separated from adjacent groups by 0-20 amino acid residues.
The nucleic acid is a polynucleotide that can be either DNA or RNA.
The invention is directed to a replicable cloning or expression vehicle comprising the above described polynucleotide sequence. The invention is also directed to such vehicle which is a plasmid. The invention is further directed to a host cell transformed with the above described DNA. The host cell can be a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line. A preferred host is Pichia pastoris. 
The invention is directed to a method of producing a single-chain antigen-binding polypeptide capable of delivering nucleic acids to a cell, comprising:
(a) providing a first genetic sequence encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) providing a second genetic sequence encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) linking the first and second genetic sequences (a) and (b) with a third genetic sequence encoding a peptide linker into a fourth genetic sequence encoding a single chain polypeptide having an antigen binding site,
wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b) or both, the single-chain antigen-binding polypeptide has an amount of basic amino acid residues sufficient to bind nucleic acids, wherein the basic amino acid residues are selected from the group consisting of: Lys, Arg and a combination thereof; and
wherein the basic amino acid residues binds nucleic acid and wherein the single-chain antigen-binding polypeptide binds antigen;
(d) transforming a host cell with the fourth genetic sequence encoding a single-chain antigen-binding polypeptide of (c); and
(e) expressing the single-chain antigen-binding polypeptide of (c) in the host, thereby producing a single-chain antigen-binding polypeptide capable of delivering nucleic acids to a cell.
The invention is further directed to a multivalent single-chain antigen-binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:
(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and
(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b) or both, the single-chain antigen-binding polypeptide has an amount of basic amino acid residues sufficient to bind nucleic acids, wherein the basic amino acid residues are selected from the group consisting of: Lys, Arg and a combination thereof; and
wherein the basic amino acid residues binds nucleic acid and wherein the single-chain antigen-binding polypeptide binds antigen.
In the above described embodiments of the invention, a lysine rich or an oligo-Lys polypeptide sequence of the present invention can be capable of attaching a polyalkylene oxide moiety wherein the polyalkylene oxide conjugated oligo-lysine tailed single-chain antigen-binding polypeptide binds an antigen as well as nucleic acids.
In the above described embodiments of the invention, the C-terminus of the second polypeptide (b) can be the native C-terminus. The C-terminus of the second polypeptide (b) can comprise a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polypeptide to be capable of binding an antigen. The C-terminus of the second polypeptide can comprise an addition of one or plurality of amino acid residue(s), such that the polypeptide is capable of binding an antigen. Moreover, the nucleic acid binding region can be generated by mutating one or a plurality of amino acid residue(s) to a basic amino acid residue(s) in the C-terminal or N-terminal regions of the polypeptide (a) or (b). In addition, the nucleic acid binding region can be generated by inserting blocks of basic amino acids at the C-terminus or N-terminus of the polypeptide (a) or (b).
In a preferred embodiment of the invention, the first polypeptide (a) can comprise the antigen binding portion of the variable region of an antibody light chain and the second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.
The invention is also directed to a method for treating a targeted disease, comprising administering an effective amount of a composition comprising a nucleic acid molecule bound to the polypeptide or protein of the invention and a pharmaceutically acceptable carrier vehicle for delivery to a cell.